1. Field of the Invention
This invention relates to modified electrodes for analysis of binding pair interactions and the use of these electrodes, especially in nucleic acid analysis and protein-protein interactions.
2. Description of the Related Art
The present invention relates to electrodes for detecting interactions between members of a binding pair, which electrodes have been modified by formation of a non-conductive self-assembled monolayer, and to the method of detecting biomolecules, such as nucleic acids or other targets, including receptors, ligands, antigens or antibodies, utilizing such electrodes.
The detection of nucleic acid hybridization at solid surfaces has been used for the identification of infectious organisms in clinical specimens (Spargo, C. A. et al., 1993, Molecular and Cellular Probes 7, 395-404; Martin, W. J., 1994, Infectious Diseases, In The Polymerase Chain Reaction (K. B. Mullis, F. Ferre and R. A. Gibbs, eds.), pp. 406-417, Berkhauser, Boston), the quantitation of mRNA for gene expression analysis (Schena, M., et al., 1995,. Science 270, 467-470), and the sequencing or resequencing of genomic DNA on high-density "chip" arrays (Chee, M., et al., 1996, Science 274, 610-613). The disclosures of the publications and patent applications referred to herein are incorporated herein by reference. Presently, this detection involves the attachment of a fluorescent label to the target nucleic acid, which is then hybridized with a probe-modified surface and detected after washing the unhybridized DNA away from the solid surface. Since detection of photons is required for detection of hybridization, analysis of arrays labeled in this manner requires high-resolution fluorescence microscopes. Alternatively, indirect detection of hybridization can be accomplished using sandwich assays where the surface-bound hybrid is subsequently hybridized to an additional signal probe that carries one or more fluorescent labels or enzymes that convert a non-fluorescent substrate to a fluorescent one (Spargo, C. A. et al., 1993, Molecular and Cellular Probes 7, 395-404). By attaching multiple enzymes to the signal probes, large signal amplification can be achieved (Holodniy, M. et al., 1995, J. Virology 69, 3510-3516); however, the preparation of these multiple enzyme systems is complex.
Other workers have developed a gene detection method utilizing a nucleic acid probe immobilized on a carrier and a specific recognizing substance for double-stranded nucleic acid, but these methods do not allow recognition of single-stranded targets because intercalation of the reporter group in the nucleic acid is required (Hashimoto et al., U.S. Pat. No. 5,776,672).
The patents of Heller (U.S. Pat. Nos. 5,532,129; 5,565,322; 5,605,662; and 5,632,957) disclose the use of an electrode with a permeation layer which is an agarose gel placed on the electrode. Application of a potential to the electrode brings probe or target nucleic acid to the reaction site on the electrode but is not part of the detection step which proceeds via use of fluorescent probes.
Organosilanes may be covalently attached at selected positions of a hydroxylated surface of a substrate, such as silicon dioxide, to form an organosilane monolayer or bilayer film or coating, as set forth in the patent of Chrisey et al. (U.S. Pat. No. 5,688,642). Organosilanes are used that have at least one reactive site for binding to the hydroxylated surface of the substrate and another reactive site that is incapable of binding either to other organosilane molecules of the coating or to the substrate, but is available for binding to a molecule distinct from these, such as a nucleic acid modified by the addition of a thiol or amino group.
Labeled proteins and soluble reagents have been used to detect protein-protein interactions. For example, the patent of Weetall (U.S. Pat. No. 5,066,372) discloses a support layer on a working electrode that is porous to reagents and to which protein can be immobilized. See also U.S. Pat. Nos. 4,945,045 of Hill, 4,545,382 of Higgins, and 5,378,628 of Gratzel.
The paper of Wang et al. (Wang et al., 1997, Anal. Chem. 69, 4056-4059), describes a membrane-covered carbon electrode for analysis of oligonucleotides in the presence of polymeric nucleic acids. The purpose of the membrane is to exclude the polymeric DNA, while small molecules can pass through the membrane for electroanalysis by the carbon electrode. The membrane is not used for attachment of probes and the membrane-covered electrodes do not offer discrimination at the sequence level.
The parent applications, whose entire specifications, drawings, and claims are specifically incorporated herein by reference, disclose, among other inventions, sequencing and methods of qualitatively and quantitatively detecting nucleic acid hybridization. Such inventions represent a major advance in the art and provide oxidation-reduction complexes which function in a catalytic manner without the addition of an enzyme or fluorescent label, provide for a catalytic current to give the concentration of guanine, or alternate base, in a manner useful for determining the presence or absence of a target nucleic acid, and provide for extremely accurate testing.
The formation of self-assembled monolayers on surfaces has enabled the design of new interfaces for the study of specific redox-active analytes, solar energy conversion and fundamental electrochemistry. Prior monolayers have been formed via alkanethiol-gold linkage and related linkages between carboxylates and phosphonates and metal oxide surfaces, such as tin-doped indium oxide. Thus, self-assembly has been used to control the structure of oligomeric DNA monolayers on gold in high salt concentrations with DNA functionalized at the 5' end with a thiol group connected to the oligonucleotide by a hexamethylene linker. The DNA apparently remains attached through its thiol end group while contacts between DNA backbones and the surface are prevented by the formation of a mercaptohexanol monolayer. The oligomeric nucleic acid probe readily hybridizes to its complementary sequence. (Levicky, R. et al., 1998, J. Amer. Chem. Soc., 120, 9787). Other systems that have been designed utilizing direct electron transfer from nucleic acids which have been contacted with an electrode, but do not use mediated electron transfer nor a self-assembled monolayer include those of Hall et al., PCT/GB93/00631.
For use in surface modification of wide-bandgap semiconductors or for interrogating interfacial electron-transfer reaction kinetics, carboxylate-functionalized ruthenium bipyridyl complexes may be used together with high-area nanocrystalline titanium dioxide films as one way to obtain surface attachment. Another way to accomplish surface attachment to nanocrystalline TiO.sub.2 in film (electrode) or colloidal form, and for subsequent retention of the molecule over a wide pH range is hexaphosphonation of Ru(bpy).sub.3.sup.2+ (Yan, S. G. et al., 1996, J. Physical Chem., 100, 6867). This prior technique does not relate to mediated solution electrochemistry as in the current invention, but rather relates to direct electron transfer, using light as a stimulus instead of a voltage.
Prior work with self-assembled monolayers has included formation of monolayers terminated by constituents such as methyl or hydroxide to which members of binding pairs could not be bound and which are used for purposes different from, and generally inconsistent with, the binding of biomolecules to the monolayers. For example, self-assembled monolayers of long-chain alkanehydroxamic acids adsorbed on metal oxides, and terminated by methyl or hydroxyl, have been used for corrosion inhibition on the metals (Folkers, J. P. et al., 1995, Langmuir, 11, 813 and Laibinis, P. E. et al., 1989, Science, 245, 845) and self-assembled thiol-terminated monolayers have been formed that bind metals electrostatically (Tarlov, M. J. and Bowden, E. F., 1991, J. Am. Chem. Soc., 113, 1847).
Early work related to the invention described herein was done with the formation of monolayers of 1,12-dodecanedicarboxylic acid (DDCA) on indium tin oxide (ITO) electrodes, with the electrodes being further derivatized with DNA via reaction of the pendant carboxylate with endogenous amines of the nucleobases following activation with water-soluble carbodiimide (Napier, M. et al., 1997, Langmuir, 13, 6342). The attachment of DNA to the electrode leads to a large catalytic enhancement due to the oxidation of guanine by the oxidized metal complex Ru(bpy).sub.3.sup.3+. The carboxylate-ITO interface is compatible with the electrochemistry of Ru(bpy).sub.3.sup.2+/3+ at E.sub.1/2 =1.05 V (vs Ag/AgCl), which would not be the case with gold-thiol monolayers. However, the 1,12 dodecane dicarboxylic acid monolayer is not stable under thermal stress, and, compared to the phosphonate of the invention herein, the carboxylate monolayer does not form reproducibly due to its lower stability.
Prior to the invention herein, self-assembled monolayers had not been described that allowed for straightforward attachment of oligonucleotide probes and the electrochemical detection of immobilized DNA via guanine oxidation. The self-assembled monolayers of this invention are thermally stable, oxidation resistant, and are formed rapidly and reproducibly. When carboxylate is used as the terminal group, nonspecific binding is minimized. Furthermore, the preferred phosphonate compounds which are used in the invention were previously unavailable or very difficult to synthesize (with only the C.sub.3 carboxy phosphonate and the C.sub.3 amino phosphonate being known to be commercially available).
Prior work with other phosphonate compounds has been in solution, for example, to enhance chromatographic separation (Lukes, I. et al., 1994, J. Am. Chem. Soc., 116, 1737), to form insulating multilayer films (e.g., thiol phosphonate in Hong, H-G, et al., 1991, Langmuir, 7, 2362, and metal alkanebisphosphonate in Yang, H. C. et al., 1993, J. Am. Chem. Soc., 115, 11855) or insulating monolayers (Kayyem, J. et al., PCT/US97/20014, to provide a passivation agent on the electrode surface that distances the oligonucleotide from the electrode, keeps charge carriers away from the surface of the electrode and blocks solvent accessibility to the electrode so that electron transfer only occurs at desired locations), or to study the reaction of phosphonic acid with a metal surface (Gao, W. et al., 1996, Langmuir, 12, 6429). Systems for orthogonal self-assembly of functionalized thiols, carboxylic acids or phosphonic acid, tagged, for example, with ferrocene were studied by Gardner, T. J. et al., 1995, J. Am. Chem. Soc., 117, 6927. None of this work relates to binding of a member of a binding pair to a self-assembled phosphonate monolayer on an electrode. Where DNA has been immobilized on such a prior film, it has been via intercalation of the DNA in double-stranded DNA by electrostatic binding and not by covalent attachment (Xu, X-H et al., 1994, J. Am. Chem. Soc., 116, 8386).
Other work with layers on electrodes relates not to monolayers but either to bilayers, for example, investigating lipid containing bilayers assembled on SiO.sub.2 and the interactions of ligands with biomolecules (Boxer et al., PCT/US97/21835), or bilayers having a space between the membrane and the electrode used to detect selected nucleic acid sequences (Harding et al., PCT/AU97/00316); to polymerized layers (Ribi (EP 0 402 917 B1) using biosensors employing electrical, optical and mechanical signals having an electrically conductive surfactant layer to which are bound members of a specific binding pair, which may be present as a uniformly oriented layer that is electrically conducting as a result of polymerization of polyunsaturated groups in the surfactant film, formed by standard lipid monolayer technologies; or to semi-permeable membranes used for entirely different purposes (Maley et al.,U.S. Pat. No. 5,711,868, in which an electrochemical sensor is used for sensing glucose by an enzyme in which the working electrode is covered with a semi-permeable membrane).
It is therefore an object of the invention to provide a method of immobilizing an oligonucleotide probe or protein-binding substance on the surface of an electrode, such as ITO, so that they are available for hybridization to a target nucleic acid or binding to a target protein, and subsequent detection via an oxidation-reduction reaction.
It is a further object of the invention to provide a method of making a non-conductive self-assembled monolayer on an electrode that may be used for the detection and quantitation of target biomolecules, such as nucleic acids or other targets, including receptors, ligands, antigens or antibodies.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.